The kidney plays an essential role in the maintenance of the volume and composition of extracellular fluid (ECF); a function which is essential for life. The kidney accomplishes this role by means of specific transport proteins in individual cell membranes of renal tubules which facilitate and in some cases promote the movement of solutes and water across cell membranes. These transport proteins, which are carefully modulated by the kidney in response to changes in ECF composition and volume are key factors in determining the quantities of solutes and water excreted from the body. The significance of the proposed research is that it is focused on expanding our knowledge concerning the mechanisms responsible for ion transport in the kidney, their role in the regulation of cell volume, and precisely, how the transporters are regulated. With this information we will be better able to understand how the normal kidney functions, and to identify ways of correcting renal dysfunction which lead to abnormalities in ECF volume and composition such as hyper- and hypokalemia, disorders of acid-base balance and hypertension. The proposed research is focused specifically on the study of chloride and potassium transport in the proximal tubule and in the diluting segment (e.g. thick ascending limb). The experiments will assess the function of nephron segments isolated and perfused in vitro and of thick ascending limb cells grown in cell cultures. The methodology includes ion selective and conventional microelectrode techniques to identify conductive and electroneutral transport pathways, and to define precisely electrochemical gradients across cell membranes, video-optical techniques to examine cell volume regulation, and the patch clamp technique to assess the presence, the role and the characteristics of ion channels in these nephron segments.